The focus of the research program is the development, evaluation, and standardization of methods to assess the quality of vaccine products and to study the immune response to bacterial vaccines. Specific projects are aimed at the development of new bioassays or the adaptation of research-based methods, leading to the availability of appropriately validated and standardized procedures that can be applied in a meaningful way either to monitor the safety, potency, and consistency of a product or to assess the immune response to infection or immunization. The emphasis of the program is the methodology rather than a specific product, and, as a result, projects involve different bacterial vaccines. Current activities involve pertussis, anthrax, and diphtheria vaccines. Many of the methods are intended to be used in support of regulatory decisions, therefore, during the methods development process, needs for reference materials are identified and, when appropriate, production and standardization of materials is coordinated. Additionally, well-designed bioassays require the efficient utilization of information through appropriate statistical analyses. As a result, collaborations with statisticians have been established to develop procedures to optimize the evaluation of bioassay data. Serologic studies of host response to pertussis vaccines and infection. Three DTaP vaccines containing an acellular pertussis component are currently being used in the U.S. for the primary series in infants; additional DTaP-based combination products for primary immunization of infants are under development and are undergoing clinical evaluation. Other acellular pertussis vaccines are under active investigation as boosters in adults and adolescents. Human immunogenicity trials are integral to all of the products under development. In the absence of a laboratory correlate of protection, serologic assay will be the principal method used by manufacturers and regulatory agencies to assess vaccine immunogenicity and will influence licensing decisions for these products. Because the trials are being conducted in many different countries and because sera are being assayed in laboratories throughout the world, international standardization of these assays is important to the interpretation and evaluation of results. Additionally, immunoassays have been shown to be helpful in the confirmation of pertussis infections in adolescents and adults. Investigation of outbreaks and implementation of effective control measures have been hampered by a lack of readily available tests to confirm suspected cases in this age group. Aims of the project include: a) to work toward the international standardization of immunoassays used to evaluate the antigen and isotype specific responses to Bordetella pertussis; b) to employ these assays to evaluate the serologic response in individuals immunized with pertussis vaccine; and c) to improve the immunoassays used to confirm pertussis infections in adolescents and adults. FY03 activities: 1) Completed evaluation of the data from a previously published international collaborative study that evaluated immunoassays used in acellular pertussis vaccine efficacy and immunogenicity studies. The data were used to evaluate a statistical model that related the coefficient of variation and the probability of observing a k-fold difference among repeated measurements. 2) Participated in analysis of data generated from a CDC-funded contract entitled: "Measurement of antibodies to three antigens of B. pertussis." The goal of the study is to develop pertussis serodiagnostic assays that can be transferred to state and regional clinical laboratories. Aided by an inter-agency agreement with the National Immunization Program of the CDC, the laboratory is initiating the laboratory phase of a collaborative project to develop and evaluate a simple, readily transferable immunoassay that can be used to confirm pertussis in adolescents and adults. 3) Initiated a collaborative project with a WHO working group to prepare and test a candidate international reference serum. Studies on Anthrax Vaccines. Anthrax is considered a major bioterrorism threat because the spores of Bacillus anthracis are very hardy, easy to disperse, and potentially easy to obtain; anthrax vaccination is considered a critical component of the defense against this agent. Currently, there is one US-licensed anthrax vaccine and new vaccines are under development. The major antigen in this preparation is believed to be a protein known as protective antigen (PA) and research in animal models has indicated that antibodies to PA can protect against B. anthracis. Efforts are under way to develop and standardize improved quality control tests for anthrax vaccines. Improved laboratory tests to assess the safety, purity, and potency of anthrax vaccines will enhance the control of the licensed product and expedite licensure of new generation immunogens. Currently available methods assess the potency of anthrax vaccines by evaluating protection from a lethal challenge with virulent spores; the goals of the project are to develop non-lethal animal methods that do not require viable bacteria, and even in vitro methods that eliminate the need for laboratory animals. FY 2003 activities: 1) Development and evaluation of a non-lethal mouse model in which the immune response to specific components of the vaccine is measured continues. An ELISA to measure anti-PA has undergone extensive validation. A second ELISA to measure anti-LF antibodies in mouse serum is being developed. 2) An essential component of these activities is the production and eventual distribution of appropriate reference reagents with the goal of international standardization of the assays. To date, two mouse immune globulin preparations enriched in anti-PA antibodies have been obtained and freeze-dried. A mouse immune globulin preparation enriched in anti-LF antibodies has been obtained and will be subject of freeze-drying shortly. 3) Immunogenicity in mice of rPA in five different antigen-adjuvant formulations was assessed, including aluminum phosphate and aluminum hydroxide as adjuvants. Results indicated that aluminum phosphate adjuvant is able to significantly increase the immune response to rPA irrespective of its adsorption to the adjuvant. Additionally, findings suggested that comparable immune responses to rPA can be obtained with both adjuvants; however the amount of adjuvant has an influence on the antibody response. 4) Continued development of quantitative and robust purity assays for in-process testing of intermediate components using HPLC and LC-MS. Additionally, these developmental methods have been applied to characterization of anthrax antigens for a reagent repository. Development of methods for evaluation of bacterial vaccines. A critical component of a biologics license application is the description of the validation of analytical methods and the establishment of specifications for the product subject of the application and its intermediates obtained during manufacturing. Assessment of vaccine quality relies heavily on the quality of the analytical tools used to evaluate the characteristics of the final bulk product and intermediaries during its manufacturing. The laboratory works with other laboratories within OVRR and with sponsors to translate scientific findings into practical methods and to ensure the timely development of standardized tests for the quality assessment of bacterial products. Additionally, during the methods development process, needs for reference materials are identified and production and standardization of materials is coordinated. FY2003 activities: 1) Evaluations of diphtheria toxoid vaccines are based on measurement of the toxin-neutralizing serum antibodies. Toxin-neutralizing antibodies are being measured in sera of guinea pig immunized with diphtheria toxoid vaccines using a Vero cell assay using low amounts of toxin and an in vivo guinea-pig skin neutralization (Lr) test. Comparison of results of diphtheria antitoxin measurement in guinea pig sera, using a lethal (L+) and a non-lethal (Lr) system has been performed in order to evaluate alternatives to the current lethal challenge test. 2) Animal models are used for the evaluation of the safety and immunogenicity of acellular pertussis vaccines before administration to humans. The mouse aerosol challenge model provides a reproducible system for the study of virulence factors and immunity involved in respiratory infection and subsequent disease, however, there is no laboratory correlate of vaccine-mediated protection. Previously, our laboratory participated in an international, multi-center collaborative study that evaluated the intranasal model using vaccines of varying potency. In FY2003, we participated in a WHO-sponsored meeting to discuss the data, draft a report, and recommend further action. Animal Model for Gastrointestinal Anthrax. Led by Noreen Hynes. The goal of the project is the development of an animal model to examine vaccine efficacy in preventing gastrointestinal anthrax following oral challenge. Gastrointestinal anthrax is the most common form of naturally-occurring human anthrax worldwide usually following the ingestion of contaminated meat from infected cattle. However, deliberate contamination of food after it is cooked, before cooking if a non-sporocidal temperature is used, or uncooked (raw) foods could result in gastrointestinal anthrax. This latter threat demands an examination of the efficacy of existing and future anthrax vaccines against ingestion-associated disease. To address this need, the Laboratory has outlined a research program with the following aims: a) to define a small animal model for gastrointestinal anthrax including the initiation of infection and the role of gut-associated dendritic cells in pathology and protection, b) to determine the protective efficacy of pre-exposure and post-exposure immunization against oral challenge, c) to characterize the immune response to sublethal oral anthrax challenge. FY2003 activities: 1) Evaluation of past published research examining animal response to oral anthrax challenge; 2) identification of potential small animal models for use; and 3) evaluation of spore preparation techniques for oral delivery. Development, Standardization, and Application of Immunological Methods to Evaluate Immune Response to Anthrax Vaccines. Led by Noreen Hynes. Although naturally-occurring human anthrax is uncommon in the U.S., the anthrax attacks in the fall of 2001 using letters as weapons proved that we need to be prepared to meet the medical and public health challenges of a future anthrax attack. Currently, there is one licensed anthrax vaccine. It is available almost exclusively in military personnel. Newer vaccines are in development for civilian use. The rarity of human anthrax cases means that animals will need to be used to judge the efficacy of these newer vaccines. Therefore, standardized serological assays are needed to assess and compare the immune response to these vaccines in both animals and humans and to compare responses to different vaccines in development. This research project focuses on the optimization and standardization of immunological assays to assess the immune response to anthrax vaccines. Assays that measure functional antibodies in serum and the intestines are emphasized. To address this need, the Laboratory has outlined a research program with the following aims: a) to evaluate and standardize assays that can be used to evaluate the immune response in humans and relevant animal models; b) to standardize existing cell-culture-based serological assays that measure functional antibodies to the major immunogenic protein of Bacillus anthracis, protective antigen, for use in both animals and humans vaccine studies; c) to minimize the use of non-biodegradable or potentially carcinogenic reagents in such assays; and d) to develop serological and tissue-based assays for assessment of secretory immunoglobulin A to support gastrointestinal anthrax animal model development. FY 2003 activities: 1) Initial evaluation of the murine macrophage-like cell line, RAW264.7, as an alternative to J774A.1 cells for use in an anthrax lethal toxin neutralization assay. 2) Initial assessment of impact of cryoprotective agents on post-thaw growth characteristics and cell sensitivity to lethal toxin in the cell-based toxin neutralization assay. 3) Performed toxin neutralization assays to support rPA vaccine development studies, to characterize anti-PA reference sera, and to assist OBRR scientists with testing associated with production of specific immunoglobulins. 4) Participated in inter-laboratory collaborative study coordinated by the CDC to assess the robustness of the human anti-PA IgG ELISA. Each of three analysts performed three independent assays on each of 16 test sera provided by the study coordinators. Determined intra-laboratory precision of the method and demonstrated comparability of commercially available software to CDCs program for calculating results. This project incorporates FY2002 projects 1Z01BJ007001-09, 1Z01BJ007003-19, 1Z01BJ007004-09, and 1Z01BJ007005-02.